Soybean markers linked to scn resistance

ABSTRACT

This disclosure concerns compositions and methods for identifying the SCN resistant phenotype in soybean. In some embodiments, the disclosure concerns methods for performing marker-assisted breeding and selection of plants carrying one or more determinants of SCN resistance in soybean.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/287,848 filed Nov. 2, 2011 which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/410,783, filed Nov. 5, 2010,the disclosure of which is hereby incorporated herein in its entirety bythis reference.

TECHNICAL FIELD

The present disclosure relates to plant disease resistance. In someembodiments, the disclosure relates to Soybean cyst nematode (SCN)resistance in soybean. In particular embodiments, the disclosure relatesto compositions and methods for identifying an SCN resistance trait inan organism, for example, molecular markers that are tightly linked toSCN resistance. Further embodiments relate to compositions and methodsfor introducing an SCN resistance trait into a host organism, forexample, by using molecular markers tightly linked to SCN resistance.

BACKGROUND

The soybean, Glycine max, is one of the major economic crops grownworldwide as a primary source of vegetable oil and protein. Growingdemand for low cholesterol and high fiber diets has increased soybean'simportance as a food. Over 10,000 soybean varieties have now beenintroduced into the United States, of which a limited number form thegenetic base of cultivars developed from hybridization and selectionprograms. Johnson and Bernard, The Soybean, Norman Ed., Academic Press,N.Y., pp. 1-73, 1963.

Soybean cyst nematode, (SCN, Heterodera glycines (HG) Ichinohe) is thesingle most damaging pest affecting soybean in the U.S. as well as inmost of the other top soybean-producing countries of the world. Theestimated yield reduction in the United States was between approximately2.9 and 3.4 million tons in 2003 and 2004, which resulted in anestimated annual loss of approximately $1.5 billion. Wrather et al.(2001); Wrather and Koenning (2006). The SCN phenotype is a very complextrait, which is controlled by multiple genes, both recessive anddominant. Concibido et al. (2004). SCN phenotyping is time consuming,cost and labor intensive.

SCN infection causes various symptoms that may include chlorosis of theleaves and stems, root necrosis, loss in seed yield, and suppression ofroot and shoot growth. The aboveground symptoms of SCN infection are notunique to SCN infection, and could be confused with nutrient deficiency,particularly iron deficiency, stress from drought, herbicide injury oranother disease. The first signs of infection are groups of plants withyellowing leaves that have stunted growth. The pathogen may also bedifficult to detect on the roots, since stunted roots are also a commonsymptom of stress or plant disease. Adult females and cysts of SCN areabout 1/32 inch long and, thus, visible without magnification.Observation of adult females and cysts on the roots is the only accurateway to detect and diagnose SCN infection in the field.

The presence of SCN is usually not obvious at the time of initial soilinfestation. The SCN population density must increase in the soil untilit is sufficient to cause above-ground symptoms on plants or a decreasein yield. Population densities may take several years to reachsignificant numbers. Thus, current SCN damage is the result ofinfestations that have been growing for several years. Although soybeanis the primary host of SCN, other legumes can serve as hosts, forexample: green beans, snap beans, dry beans, red beans, lima beans, mungbeans, bush beans, Adzuki beans, garden peas, and cowpeas. There arethirty days in the SCN life cycle. Thus, a single growing seasonencompasses multiple generations of the parasite. Moreover, SCN eggs mayremain intact in soil for several years before hatching.

In the past, an SCN population was given a “race” designation bycomparing its reproduction on a set of four soybean germplasm lines withthat on a standard SCN-susceptible soybean cultivar. The most commonlyused race scheme identified 16 races of SCN. The race designationallowed nematologists and soybean breeders to share information aboutthe ability of certain SCN populations to reproduce on soybean varietiesthat contain certain genes for resistance to SCN.

In 2003, the HG Type Test was developed to replace the race test. Thisnew test includes seven sources of resistance (germplasm lines) and theresults are shown as a percentage, indicating how much the nematodepopulation from a soil sample increased on each of the seven lines. Thistest indicates which sources of resistance would be good for aparticular field being tested, and which would be poor. Since thegenetic sources of resistance are currently limited in commerciallyavailable soybean varieties, it is important to rotate these “sources ofresistance” to delay the build-up of a virulent SCN population.

Shortly after the discovery of SCN in the United States, sources of SCNresistance were identified. Ross and Brim (1957) Plant Dis. Rep.41:923-4. Some lines, such as Peking and PI 88788, were quicklyincorporated into breeding programs. Peking became widely used as asource of resistance due to its lack of agronomically undesirabletraits, with Pickett as the first SCN resistant cultivar released. Therecognition that certain SCN resistant populations could overcomeresistant cultivars led to an extensive screen for additional sources ofSCN resistance. PI 88788 emerged as a popular source of race 3 and 4resistance, even though it had a cyst index greater than 10% (but lessthan 20%) against race 4, and Peking and its derivatives emerged as apopular source for races 1 and 3. PI 437654 was subsequently identifiedas having resistance to all known races and its SCN resistance wasbackcrossed into Forrest. Currently, there are more than 130 PIs knownto have SCN resistance. PI 209332 and PI 90763 are other exemplary SCNresistant soybean breeding lines. Not all varieties with the same sourceof resistance have comparable yields, nor do they respond identically toSCN.

Resistant soybean varieties are the most effective tool available formanagement of SCN. SCN densities usually decrease when resistantsoybeans are grown because most SCN juveniles are unable to feed anddevelop on the roots of the resistant varieties. However, in anynaturally infested field, a few SCN juveniles (<1%) will be able toreproduce on the resistant varieties currently available. The number ofSCN juveniles that can reproduce on resistant soybean varieties canincrease when resistant varieties are grown repeatedly. Eventually, theSCN population may be able to reproduce as well on a resistant varietyas a susceptible variety if SCN-resistant soybeans are grown every timesoybeans are produced in an infested field. Fortunately, the number ofSCN juveniles that can reproduce on resistant varieties declines whensusceptible soybean varieties are grown because these nematodes do notcompete well for food with the other SCN juveniles in the soil thatcannot feed on the resistant varieties.

SCN race 3 is considered to be the most prominent race in the Midwesternsoybean producing states. Considerable effort has been devoted to thegenetics and breeding for resistance to race 3. While both Peking and PI88788 are resistant to SCN race 3, classical genetics studies suggestthat they harbor different genes for race 3 resistance. Rao-Arelli andAnand (1988) Crop Sci. 28:650-2. Race 3 resistance is probably under thecontrol of three or four different genes. Id.; see also Mansur et al.(1993) Crop Sci. 33:1249-53. One major SCN resistance QTL that maps tolinkage group G is rhgl. Concibido et al. (1996) Theor. Appl. Genet.93:234-41. Other SCN resistance QTLs map to linkage groups A2, C1, M, D,J, L25, L26, and K. Id.; U.S. Pat. No. 5,491,081. SCN resistance QTLsbehave in a race-specific manner, at least by accounting for differentproportions of the total phenotypic variation with respect to differentSCN races. Concibido et al. (1997) Crop Sci. 37:258-64. However, therhgl locus on linkage group G may be necessary for the development ofresistance to any of the identified SCN races. But see Qui et al. (1999)Theor. Appl. Genet. 98:356-64.

Markers that are linked to SCN traits include RFLPs, SSRs and SNPs. TheSNP markers identified in this disclosure can be used to do SCNgenotyping to support a breeding program. Using the presently disclosedSNP markers to perform SCN genotyping in support of a breeding programprovides: cost and time savings, early selection of desired progeny, andmore accurate and rapid commercialization of SCN resistant soybeanvarieties.

DISCLOSURE

Molecular markers that are linked to an SCN phenotype may be used tofacilitate marker-assisted selection for the SCN resistance trait insoybean. Marker-assisted selection provides significant advantages withrespect to time, cost, and labor, when compared to SCN phenotyping.Surprisingly, it is disclosed herein that among 15 SNP markersidentified to be within or near SCN disease resistance QTL regions inthe soybean genome that were polymorphic in parent genotypes, only threewere linked to the SCN resistance trait. These three SNP markers, then,offer superior utility in marker-assisted selection of SCN resistantsoybean varieties.

Described herein are nucleic acid molecular markers that are linked to(e.g., linked, tightly linked, or extremely tightly linked) an SCNresistance phenotype. In particular examples, the molecular markers maybe SNP markers. Also described herein are methods of using nucleic acidmolecular markers that are linked to an SCN resistance phenotype, forexample and without limitation, to identify plants with an SCNresistance phenotype, to introduce an SCN resistance phenotype into newplant genotypes (e.g., through marker-assisted breeding or genetictransformation), and to cultivate plants that are likely to have an SCNresistance phenotype.

Further described are means for introducing an SCN phenotype to soybeanand means for identifying plants having an SCN phenotype. In someexamples, a means for introducing an SCN phenotype to soybean may be amarker that is linked (e.g., linked, tightly linked, or extremelytightly linked) to an SCN phenotype. In some examples, a means foridentifying plants having an SCN phenotype may be a probe thatspecifically hybridizes to a marker that is linked (e.g., linked,tightly linked, or extremely tightly linked) to an SCN phenotype.

Also described herein are plants and plant materials that are derivedfrom plants having an SCN phenotype as identified using molecularmarkers described herein. Thus, soybean plants that are produced bymarker-assisted selection using one or more molecular marker(s) that arelinked to an SCN resistance phenotype are described.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a through 1c include a list of QTLs associated with SCNresistance that have been reported in the SCN literature.

FIGS. 2a thorugh 2 b include a representation of the soybean genome,including chromosomes and linkage groups (LGs).

FIG. 3 includes a representation of soybean chromosome 18 (linkage groupG), and QTLs and QTL intervals associated with SCN resistance and SNPslocated therein.

FIG. 4 includes a representation of soybean chromosome 8 (linkage groupA2), and QTLs and QTL intervals associated with SCN resistance and SNPslocated therein.

FIG. 5 includes a representation of soybean chromosome 11 (linkage groupBi), and QTLs and QTL intervals associated with SCN resistance and SNPslocated therein.

FIG. 6 includes a representation of soybean chromosome 20 (linkage groupI), and QTLs and QTL intervals associated with SCN resistance and SNPslocated therein.

FIG. 7 includes clusters of 24 soybean SCN related cultivars or parentallines on four SNP loci. Also included is a table showing the 24 soybeancultivars and SCN mapping parents used. In the table, the first row ofsamples and the last two samples in the second row were SCN susceptible(green), and the first ten samples in the second row were SCN resistant(yellow). The last three samples in the second row were parental linesof two SCN mapping populations.

FIG. 8 includes clusters of 96 lines on three SNPs loci that showedco-segregation with the SCN resistance trait.

FIG. 9 includes the distribution of the SCN indexes assigned to mappingpopulations. The histogram shows a range from 0.01 to 3.8, with a meanof 0.63, and a median of 0.465.

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases, asdefined in 37 C.F.R. § 1.822. Only one strand of each nucleic acidsequence is shown, but the complementary strand is understood to beincluded by any reference to the displayed strand. In the accompanyingsequence listing:

SEQ ID NO:1 shows a primer sequence used in a KBiosciences CompetitiveAllele-Specific PCR SNP genotyping system (KASPAR™) assay that isspecific for the rhgl-3995 allele:GAAGGTGACCAAGTTCATGCTGGAATTATGTTGGGTTTTTTTTCTTTCTGT.

SEQ ID NO:2 shows a second primer sequence used in a KASPAR™ assay thatis specific for the rhg1-3995 allele:GAAGGTCGGAGTCAACGGATTGAATTATGTTGGGTTTT TTTTCTTTCTGG.

SEQ ID NO:3 shows a common reverse primer sequence used in a KASPAR™assay for rhg1-3995: GCCCAGAAAAAAGGGATAAATAACGGATA.

SEQ ID NO:4 shows a primer sequence used in a KASPAR™ assay that isspecific for the NCSB_004074 allele:GAAGGTGACCAAGTTCATGCTATTATGTTGTAACACAA ATTTGCACCTCAT.

SEQ ID NO:5 shows a second primer sequence used in a KASPAR™ assay thatis specific for the NCSB_004074 allele: GAAGGTCGGAGTCAACGGATTATGTTGTAACACAAATTTGCACCTCAG.

SEQ ID NO:6 shows a common reverse primer sequence used in a KASPARTMassay for NCSB_004074: CTATACAACTAAATCGTAATTCCATTGTAT.

SEQ ID NO:7 shows a primer sequence used in a KASPAR™ assay that isspecific for the BARC_010889-01691 allele:GAAGGTGACCAAGTTCATGCTGAAAAAATAAAA TTGATCATCACATATGGTTAG.

SEQ ID NO:8 shows a second primer sequence used in a KASPAR™ assay thatis specific for the BARC_010889-01691 allele:GAAGGTCGGAGTCAACGGATTGAAAAAAT AAAATTGATCATCACATATGGTTAA.

SEQ ID NO:9 shows a common reverse primer sequence used in a KASPAR™assay for BARC_010889-01691: TAAGTGAGGGCAATGTATTAGTATYAAGTA.

SEQ ID NO:10 shows a marker NCSB_004074 sequence:

CACGATTTTGTTGTGTTACATAAATTACTATACAACTAAATCGTAATTCCATTGTATTAC[A/C]TGAGGTGCAAATTTGTGTTACAACATAATTGTAATTTTATTGTACGATAAAAACTATAAC.

SEQ ID NO:11 shows a marker BARC_010889-01691 sequence:

CTCTTCACACCTTTAAGGAAGTTAGTACCATTCCACTATTCAAGTATTTTTTTTAATTCAAAATTATTAAGTGAGGGCAATGTATTAGTATNAAGTA[C/T]TAACCATATGTGATGATCAATTTTATTTTTTCATGGCTTTGTCGAAAGTAACATTATATTGTGGTTTTAAATGAAAATCTGTGATTTGCAT.

SEQ ID NO:12 shows a marker rhg1-3995 sequence:

TCTGATAACTATGACAGCATCTTCCAAGATAATGACTTCCAAGTTCCAACACTGGCTCTGTACATTTGAACTAATTTTATATCATTTATCTATTGTGATTGAAATATAAAATTGAAGTGATGTGAACAATACAAATCACATCTTGAATTAAAATATCTAACAACTGGAACAAATAAGAGGCCCAGAAAAAAGGGATAAATAACGGATAACAAG[A/C]CAGAAAGAAAAAAAACCCAACATAATTCCAACTTCAAAATTCACTCAATAAAAAGTTTAACATGTAAATTTACTTGGAAACAAAACTCATAACCAATAATAATAATAATAAAAGAAATCAGTTTTATAGCATTAATTTGGGATGCTCTGCTTGTATGCAAATGGCACAACCTTACCCTCAAGATTGCAAAACACAGATGAGTAACAGATGCAATGTGAATCAATAAAAAGTATTGTTGCGTTGTTGATGACACAACCTTACTCATAAAAAATGCAT.

DETAILED DESCRIPTION I. Overview of Several Embodiments

Particular embodiments include three exemplary SNP markers (rhgl-3995,BARC_010889 01691, and NCSB_004074) that show co-segregation with thesoybean cyst nematode (SCN) resistance trait in 96 tested soybean lines.Markers that co-segregate with SCN resistance are linked to this traitand, therefore, may be useful in marker-assisted selection and breeding.Also disclosed herein is a strategy used to identify these threeexemplary SNP markers linked to SCN resistance. The physical mappositions of these three exemplary SNP markers in the Glycine max genomeare provided. Using the three exemplary SNP markers described herein, aspecific assay using KBiosciences Competitive Allele-Specific PCR SNPgenotyping system (KASPAR™) was developed to rapidly and accuratelyidentify plants carrying the SCN resistance trait. While embodiments ofthe invention are described with reference to three exemplary SNPmarkers linked to SCN resistance, those of skill in the art willappreciate that additional, equivalent markers may be identified usingthe techniques described herein. SNP markers linked to SCN resistancemay be used, for example, in SCN genotyping to select SCN resistantindividuals from soybean breeding populations.

Soybean cyst nematode (SCN) resistance is a very complex trait. SCNinfestation may be caused by one or more different Heterodera glycinesraces, the resistance for each of which may require different resistantgenes located on different linkage groups. See Table 1. The threemarkers disclosed in Table 1 are all located in linkage group G. The SCNresistance gene(s) in linkage group G is thought to be responsible forresistance to races 3 and 14.

The strategy described herein is used to identify markers in otherlinkage groups (for example, A₂, B₁, and I) that are linked to SCNresistance. Thus, methods for identifying such markers are alsoprovided. The general strategy is also used to map other traits ofinterest. The strategy is more efficient than traditional mappingstrategies and may be particularly useful in molecular breedingprograms.

TABLE 1 Sources of SCN resistance Resistant Germplasm H. glycines RacesLinkage groups (LG) PI 88788 3, 14 G Peking 1, 3, and 5 G, A2, and B PI437654 All G (Rhg1), A2 (Rhg4), B, C1, L25, L26, M, and D1a PI 90763 3PI 438489B 1, 2, 3, 5, and 14 G, E, A1, B1, and C1 PI 89772 1, 2, 3, and5 G, E, A1, C1, C2, and D1a PI209332 All G (Rhg1), and A2 (Rhg4) PUSCN143 A, G, B, I, and F Hartwig 3 Forrest 3 G and A2 Pyramid 3, 14 (from PI88788) A2, D, and G

II. Terms

Mapping population: As used herein, the term “mapping population” mayrefer to a plant population used for gene mapping. Mapping populationsare typically obtained from controlled crosses of parent genotypes.Decisions on the selection of parents and mating design for thedevelopment of a mapping population, and the type of markers used,depend upon the gene to be mapped, the availability of markers, and themolecular map. The parents of plants within a mapping population musthave sufficient variation for the trait(s) of interest at both thenucleic acid sequence and phenotype level. Variation of the parents'nucleic acid sequence is used to trace recombination events in theplants of the mapping population. The availability of informativepolymorphic markers is dependent upon the amount of nucleic acidsequence variation.

Backcrossing: Backcrossing methods may be used to introduce a nucleicacid sequence into plants. The backcrossing technique has been widelyused for decades to introduce new traits into plants. N. Jensen, Ed.,Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a typicalbackcross protocol, the original variety of interest (recurrent parent)is crossed to a second variety (non-recurrent parent) that carries agene of interest to be transferred. The resulting progeny from thiscross are then crossed again to the recurrent parent, and the process isrepeated until a plant is obtained wherein essentially all of thedesired morphological and physiological characteristics of the recurrentplant are recovered in the converted plant, in addition to thetransferred gene from the non-recurrent parent.

KBiosciences Competitive Allele-Specific PCR SNP genotyping system(KASPAR™): KASPAR™ is a commercially available homogeneous fluorescentsystem for determining SNP genotypes (KBiosciences Ltd., Hoddesdon, UK).A KASPAR™ assay comprises an SNP-specific “assay mix,” which containsthree unlabelled primers, and a “reaction mix,” which contains all theother required components, for example, a universal fluorescentreporting system. In addition to these mixes, the user provides, interalia, a FRET-capable plate reader, microtiter plate(s), and DNA samplesthat contain about 5 ng/L DNA.

A typical KASPAR™ assay comprises the steps of: allele-specific primerdesign (e.g., using PRIMERPICKER™, which is a free service availablethrough the internet at the KBiosciences website), preparation ofreaction mix including the allele-specific primers, admixing thereaction mix to DNA samples in a microtiter plate, thermocycling,reading the plate in a fluorescent plate reader, and plotting andscoring the fluorescent data. Data from each sample are plotted togetheron a two-dimensional graph, where the x- and y-axes correspond to FAMand VIC fluorescence values. Samples having the same SNP genotypecluster together on the plot (i.e., A/A, A/a, and a/a). More technicalinformation about the KASPar system, including a guide of solutions tocommon problems, is obtainable from KBiosciences Ltd. (e.g., the KASParSNP Genotyping System Reagent Manual).

Linked, tightly linked, and extremely tightly linked: As used herein,linkage between genes or markers may refer to the phenomenon in whichgenes or markers on a chromosome show a measurable probability of beingpassed on together to individuals in the next generation. The closer twogenes or markers are to each other, the closer to (1) this probabilitybecomes. Thus, the term “linked” may refer to one or more genes ormarkers that are passed together with a gene with a probability greaterthan 0.5 (which is expected from independent assortment wheremarkers/genes are located on different chromosomes). When the presenceof a gene contributes to a phenotype in an individual, markers that arelinked to the gene may be said to be linked to the phenotype. Thus, theterm “linked” may refer to a relationship between a marker and a gene,or between a marker and a phenotype.

Because the proximity of two genes or markers on a chromosome isdirectly related to the probability that the genes or markers will bepassed together to individuals in the next generation, the term “linked”may also refer herein to one or more genes or markers that are locatedwithin about 2.0 Mb of one another on the same chromosome. Thus, two“linked” genes or markers may be separated by about 2.1 Mb, 2.00 Mb,about 1.95 Mb, about 1.90 Mb, about 1.85 Mb, about 1.80 Mb, about 1.75Mb, about 1.70 Mb, about 1.65 Mb, about 1.60 Mb, about 1.55 Mb, about1.50 Mb, about 1.45 Mb, about 1.40 Mb, about 1.35 Mb, about 1.30 Mb,about 1.25 Mb, about 1.20 Mb, about 1.15 Mb, about 1.10 Mb, about 1.05Mb, about 1.00 Mb, about 0.95 Mb, about 0.90 Mb, about 0.85 Mb, about0.80 Mb, about 0.75 Mb, about 0.70 Mb, about 0.65 Mb, about 0.60 Mb,about 0.55 Mb, about 0.50 Mb, about 0.45 Mb, about 0.40 Mb, about 0.35Mb, about 0.30 Mb, about 0.25 Mb, about 0.20 Mb, about 0.15 Mb, about0.10 Mb, about 0.05 Mb, about 0.025 Mb, and about 0.01 Mb. Particularexamples of markers that are “linked” to the SCN phenotype in soybeaninclude nucleotide sequences on chromosome 18 of the soybean genome.

As used herein, the term “tightly linked” may refer to one or more genesor markers that are located within about 0.5 Mb of one another on thesame chromosome. Thus, two “tightly linked” genes or markers may beseparated by about 0.6 Mb, about 0.55 Mb, 0.5 Mb, about 0.45 Mb, about0.4 Mb, about 0.35 Mb, about 0.3 Mb, about 0.25 Mb, about 0.2 Mb, about0.15 Mb, about 0.1 Mb, and about 0.05 Mb.

As used herein, the term “extremely tightly linked” may refer to one ormore genes or markers that are located within about 100 kb of oneanother on the same chromosome. Thus, two “extremely tightly linked”genes or markers may be separated by about 125 kb, about 120 kb, about115 kb, about 110 kb, about 105 kb, 100 kb, about 95 kb, about 90 kb,about 85 kb, about 80 kb, about 75 kb, about 70 kb, about 65 kb, about60 kb, about 55 kb, about 50 kb, about 45 kb, about 40 kb, about 35 kb,about 30 kb, about 25 kb, about 20 kb, about 15 kb, about 10 kb, about 5kb, and about 1 kb. Particular examples of markers that are “extremelytightly linked” to the SCN phenotype in soybean include rhg1-3995,BARC_010889_01691, and NCSB_004074.

In view of the foregoing, it will be appreciated that markers linked toa particular gene or phenotype include those markers that are tightlylinked, and those markers that are extremely tightly linked, to the geneor phenotype. Linked, tightly linked, and extremely tightly geneticmarkers of the SCN phenotype may be useful in marker-assisted breedingprograms to identify SCN resistant soybean varieties, and to breed thistrait into other soybean varieties to confer SCN resistance.

Locus: As used herein, the term “locus” refers to a position on thegenome that corresponds to a measurable characteristic (e.g., a trait).An SNP locus is defined by a probe that hybridizes to DNA containedwithin the locus.

Marker: As used herein, a marker refers to a gene or nucleotide sequencethat can be used to identify plants having a particular allele. A markermay be described as a variation at a given genomic locus. A geneticmarker may be a short DNA sequence, such as a sequence surrounding asingle base-pair change (single nucleotide polymorphism, or “SNP”), or along one, for example, a microsatellite/simple sequence repeat (“SSR”).A “marker allele” refers to the version of the marker that is present ina particular individual.

The term marker as used herein may refer to a cloned segment of soybeanchromosomal DNA (for example, a segment including rhgl-3995,BARC_010889_01691, or NCSB_004074), and may also or alternatively referto a DNA molecule that is complementary to a cloned segment of soybeanchromosomal DNA (for example, DNA complementary to a segment includingrhg1-3995, BARC_010889_01691, or NCSB_004074).

In some embodiments, the presence of a marker in a plant may be detectedthrough the use of a nucleic acid probe. A probe may be a DNA moleculeor an RNA molecule. RNA probes can be synthesized by means known in theart, for example, using a DNA molecule template. A probe may contain allor a portion of the nucleotide sequence of the marker and additional,contiguous nucleotide sequence from the plant genome. This is referredto herein as a “contiguous probe.” The additional, contiguous nucleotidesequence is referred to as “upstream” or “downstream” of the originalmarker, depending on whether the contiguous nucleotide sequence from theplant chromosome is on the 5′ or the 3′ side of the original marker, asconventionally understood. As is recognized by those of ordinary skillin the art, the process of obtaining additional, contiguous nucleotidesequence for inclusion in a marker may be repeated nearly indefinitely(limited only by the length of the chromosome), thereby identifyingadditional markers along the chromosome. All above-described markers maybe used in some embodiments of the present invention.

An oligonucleotide probe sequence may be prepared synthetically or bycloning. Suitable cloning vectors are well-known to those of skill inthe art. An oligonucleotide probe may be labeled or unlabeled. A widevariety of techniques exist for labeling nucleic acid molecules,including, for example and without limitation: radiolabeling by nicktranslation, random priming, tailing with terminal deoxytransferase, orthe like, where the nucleotides employed are labeled, for example, withradioactive ³²P. Other labels which may be used include, for example andwithout limitation: Fluorophores (e.g., FAM and VIC), enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, and the like.Alternatively, the use of a label that provides a detectable signal, byitself or in conjunction with other reactive agents, may be replaced byligands to which receptors bind, where the receptors are labeled (forexample, by the above-indicated labels) to provide detectable signals,either by themselves, or in conjunction with other reagents. See, e.g.,Leary et al. (1983) Proc. Natl. Acad. Sci. USA 80:4045-9.

A probe may contain a nucleotide sequence that is not contiguous to thatof the original marker; this probe is referred to herein as a“noncontiguous probe.” The sequence of the noncontiguous probe islocated sufficiently close to the sequence of the original marker on thegenome so that the noncontiguous probe is genetically linked to the samegene or trait (e.g., SCN resistance). For example, in some embodiments,a noncontiguous probe is located within 500 kb, 450 kb, 400 kb, 350 kb,300 kb, 250 kb, 200 kb, 150 kb, 125 kb, 100 kb, 0.9 kb, 0.8 kb, 0.7 kb,0.6 kb, 0.5 kb, 0.4 kb, 0.3 kb, 0.2 kb, or 0.1 kb of the original markeron the soybean genome.

A probe may be an exact copy of a marker to be detected. A probe mayalso be a nucleic acid molecule comprising, or consisting of, anucleotide sequence which is substantially identical to a cloned segmentof the subject organism's (for example, soybean) chromosomal DNA. Asused herein, the term “substantially identical” may refer to nucleotidesequences that are more than 85% identical. For example, a substantiallyidentical nucleotide sequence may be 85.5%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to thereference sequence.

A probe may also be a nucleic acid molecule that is “specificallyhybridizable” or “specifically complementary” to an exact copy of themarker to be detected (“DNA target”). “Specifically hybridizable” and“specifically complementary” are terms that indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenthe nucleic acid molecule and the DNA target. A nucleic acid moleculeneed not be 100% complementary to its target sequence to be specificallyhybridizable. A nucleic acid molecule is specifically hybridizable whenthere is a sufficient degree of complementarity to avoid non-specificbinding of the nucleic acid to non-target sequences under conditionswhere specific binding is desired, for example, under stringenthybridization conditions.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ and/or Mg⁺ concentration) of thehybridization buffer will determine the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are known to those of ordinary skill in the art, and arediscussed, for example, in Sambrook et al. (ed.) Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames andHiggins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985.Further detailed instruction and guidance with regard to thehybridization of nucleic acids may be found, for example, in Tijssen,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” in Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2, Elsevier, N.Y., 1993; and Ausubel et al., Eds., CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, N.Y., 1995.

As used herein, “stringent conditions” encompass conditions under whichhybridization will only occur if there is less than 50% mismatch betweenthe hybridization molecule and the DNA target. “Stringent conditions”include further particular levels of stringency. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 50% sequence mismatch will not hybridize; conditions of “highstringency” are those under which sequences with more than 20% mismatchwill not hybridize; and conditions of “very high stringency” are thoseunder which sequences with more than 10% mismatch will not hybridize.

The following are representative, non-limiting hybridization conditions.

Very High Stringency (detects sequences that share at least 90% sequenceidentity): Hybridization in 5x SSC buffer at 65° C. for 16 hours; washtwice in 2× SSC buffer at room temperature for 15 minutes each; and washtwice in 0.5× SSC buffer at 65° C. for 20 minutes each.

High Stringency (detects sequences that share at least 80% sequenceidentity): Hybridization in 5×-6× SSC buffer at 65-70° C. for 16-20hours; wash twice in 2× SSC buffer at room temperature for 5-20 minuteseach; and wash twice in lx SSC buffer at 55-70° C. for 30 minutes each.

Moderate Stringency (detects sequences that share at least 50% sequenceidentity): Hybridization in 6× SSC buffer at room temperature to 55° C.for 16-20 hours; wash at least twice in 2×-3× SSC buffer at roomtemperature to 55° C. for 20-30 minutes each.

With respect to all probes discussed, supra, the probe may compriseadditional nucleic acid sequences, for example, promoters, transcriptionsignals, and/or vector sequences. Any of the probes discussed, supra,may be used to define additional markers that are tightly linked to agene involved in SCN resistance, and markers thus identified may beequivalent to exemplary markers named in the present disclosure, andthus are within the scope of the invention.

Marker-assisted breeding: As used herein, the term “marker-assistedbreeding” may refer to an approach to breeding directly for one or morecomplex traits (e.g., SCN resistance). In current practice, plantbreeders attempt to identify easily detectable traits, such as flowercolor, seed coat appearance, or isozyme variants that are linked to anagronomically desired trait. The plant breeders then follow theagronomic trait in the segregating, breeding populations by followingthe segregation of the easily detectable trait. However, there are veryfew of these linkage relationships available for use in plant breeding.

Marker-assisted breeding provides a time- and cost-efficient process forimprovement of plant varieties. Several examples of the application ofmarker-assisted breeding involve the use of isozyme markers. See, e.g.,Tanksley and Orton, eds. (1983) Isozymes in Plant Breeding and Genetics,Amsterdam: Elsevier. One example is an isozyme marker associated with agene for resistance to a nematode pest in tomato. The resistance,controlled by a gene designated Mi, is located on chromosome 6 of tomatoand is very tightly linked to Aps1, an acid phosphatase isozyme. Use ofthe Aps1 isozyme marker to indirectly select for the Mi gene providedthe advantages that segregation in a population can be determinedunequivocally with standard electrophoretic techniques; the isozymemarker can be scored in seedling tissue, obviating the need to maintainplants to maturity; and co-dominance of the isozyme marker allelesallows discrimination between homozygotes and heterozygotes. See Rick(1983) in Tanksley and Orton, supra.

Quantitative trait locus: As used herein, the term “Quantitative traitlocus” (QTL) may refer to stretches of DNA that have been identified aslikely DNA sequences (e.g., genes, non-coding sequences, and/orintergenic sequences) that underlie a quantitative trait, or phenotype,that varies in degree, and can be attributed to the interactions betweentwo or more DNA sequences (e.g., genes, non-coding sequences, and/orintergenic sequences) or their expression products and theirenvironment. Quantitative trait loci (QTLs) can be molecularlyidentified to help map regions of the genome that contain sequencesinvolved in specifying a quantitative trait.

As used herein, the term “QTL interval” may refer to stretches of DNAthat are linked to the genes that underlie the QTL trait. A QTL intervalis typically, but not necessarily, larger than the QTL itself. A QTLinterval may contain stretches of DNA that are 5′ and/or 3′ with respectto the QTL.

Sequence identity: The term “sequence identity” or “identity,” as usedherein in the context of two nucleic acid or polypeptide sequences, mayrefer to the residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.

As used herein, the term “percentage of sequence identity” may refer tothe value determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences) over a comparison window, wherein the portion ofthe sequence in the comparison window may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleotide or amino acid residue occursin both sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thecomparison window, and multiplying the result by 100 to yield thepercentage of sequence identity.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described in, for example:Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch(1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad.Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higginsand Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res.16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearsonet al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMSMicrobiol. Lett. 174:247-50. A detailed consideration of sequencealignment methods and homology calculations can be found in, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-10.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™; Altschul et al. (1990)) is available fromseveral sources, including the National Center for BiotechnologyInformation (Bethesda, MD), and on the internet, for use in connectionwith several sequence analysis programs. A description of how todetermine sequence identity using this program is available on theinternet under the “help” section for BLAST™. For comparisons of nucleicacid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn)program may be employed using the default BLOSUM62 matrix set to defaultparameters. Nucleic acid sequences with even greater similarity to thereference sequences will show increasing percentage identity whenassessed by this method.

Single-nucleotide polymorphism: As used herein, the term“single-nucleotide polymorphism” (SNP) may refer to a DNA sequencevariation occurring when a single nucleotide in the genome (or othershared sequence) differs between members of a species or pairedchromosomes in an individual. Within a population, SNPs can be assigneda minor allele frequency that is the lowest allele frequency at a locusthat is observed in a particular population. This is simply the lesserof the two allele frequencies for single-nucleotide polymorphisms.Different populations are expected to exhibit at least slightlydifferent allele frequencies. Particular populations may exhibitsignificantly different allele frequencies. In some examples, markerslinked to SCN resistance are SNP markers.

SNPs may fall within coding sequences of genes, non-coding regions ofgenes, or in the intergenic regions between genes. SNPs within a codingsequence will not necessarily change the amino acid sequence of theprotein that is produced, due to degeneracy of the genetic code. An SNPin which both forms lead to the same polypeptide sequence is termed“synonymous” (sometimes called a silent mutation). If a differentpolypeptide sequence is produced, they are termed “non-synonymous.” Anon-synonymous change may either be missense or nonsense, where amissense change results in a different amino acid, and a nonsense changeresults in a premature stop codon. SNPs that are not in protein-codingregions may still have consequences for gene splicing, transcriptionfactor binding, or the sequence of non-coding RNA. SNPs are usuallybiallelic and thus easily assayed in plants and animals. Sachidanandam(2001) Nature 409:928-33.

Trait or phenotype: The terms “trait” and “phenotype” are usedinterchangeably herein. For the purposes of the present disclosure, atrait of particular interest is SCN resistance.

III. QTL-Based Identification of Markers Linked to a Trait of Interest

A. Overview

In some embodiments, a trait (e.g., SCN resistance) is mapped using astrategy that is different from traditional mapping approaches. Forexample, a trait may be mapped according to a strategy that, for thesake of convenience, may be described as comprising four steps. In afirst step, QTL interval target regions that correspond to a trait to bemapped may be determined. In a second step, markers (e.g., SNP markers)may be selected which are located within or near determined QTLintervals of the target genome (e.g., soybean genome). In a third step,specific primers may be designed that facilitate the genotyping ofindividual subjects with respect to selected markers. In particularexamples, specific primers are designed for use in a KASPAR™ genotypingassay. In a fourth step, populations that show segregation for the traitmay be screened using the specific primers to identify those markersthat are linked to the trait.

B. Markers Linked to a Trait of Interest and the Identification Thereof

Determination of QTL interval target regions and identification ofmarkers.

QTLs may be determined by any technique available to those of skill inthe art. For example, the physical positions of a QTL that correspondsto a particular trait of interest may be initially determined byreference to the location of genes that are known to contribute to theparticular trait. In some embodiments, SCN resistance genes may beidentified on at least four regions on chromosome 8, 11, 18, and 20,respectively. See, e.g., Concibido et al. (1996) Theor. Appl. Genet.93:234-41, Concibido et al. (1997) Crop Sci. 37:258-64, Meksem et al.(1999) Theor. Appl. Genet. 99:1131-42, Qui et al. (1999) Theor. Appl.Genet. 98:356-64, Meksem et al. (2001) Mol. Breeding 7:63-71, Li et al.(2009) Mol. Breeding 24:63-76, Wu et al. (2009) Theor. Appl. Genet.118:1093-105; U.S. Pat. Nos. 5,491,081, 6,096,944, 6,162,967, 6,271,437,6,284,948, 6,300,541, 6,538,175, 7,154,021, 7,485,770; U.S.S.N.s20020129402, 20020144310, 20030005491, 20030135881, 20060225150,20060253919, 20080072352, and 20090100537; and International PCTPublication Nos. WO1995020669A2, WO2001051627A2, and WO2008153804A2. Insome embodiments, the initially identified QTLs are grouped or dividedinto a less complicated or extensive list of QTLs that may haveboundaries in the genome that are the same or different than theboundaries of the initially identified QTLs.

In some embodiments, a region of DNA may be selected that is likely tocontain markers that are linked to the QTL trait. This region may bereferred to as a QTL interval. For example, a QTL interval may be aregion of DNA that includes the QTL and additional genomic DNA that isnear the QTL in either, or both, the 5′ and 3′ directions. In someembodiments, a QTL interval may be about 4 Mb, about 3.5 Mb, about 3 Mb,about 2.5 Mb, about 2 Mb, about 1.5 Mb, or about 1 Mb.

In particular embodiments, the target genome may be searched to identifymarkers that are physically located in, near, or between the QTLs andQTL intervals. If a reference map containing the location of knownmarkers is available for the target genome, the reference map may beused to identify markers. Nucleic acid sequences of the target genomemay also be searched, for example, by software such as BLASTTM. In someembodiments, SNP markers may be identified. In some embodiments, markersmay be identified that are physically located in, near, or between QTLsand QTL intervals of the soybean genome that correspond to the SCNresistance trait. In particular examples, identified SNP markers thatare physically located in, near, or between QTLs and QTL intervals ofthe soybean genome that correspond to the SCN resistance trait may beselected from the group consisting of the markers listed in Table 2.

In other embodiments, particular markers may be selected from theidentified markers that are physically located in, near, or between QTLsand QTL intervals that correspond to a trait of interest, which markersare polymorphic among the parental lines from which a mapping populationwill be generated. Polymorphism of a given marker among the parentallines is directly related to the ability to trace recombination eventsin a mapping population produced from the parental lines.

In particular examples, polymorphic markers among parental soybean linesare selected to screen SCN resistance mapping populations to determinewhich, if any, of the polymorphic markers are linked to the SCNresistance trait. Such markers may segregate so that one allele of theSNP marker appears exclusively in SCN resistant individuals, and theother allele of the SNP marker appears exclusively in SCN susceptibleindividuals. Mapping populations may be generated by crossing onevariety that is SCN resistant with another variety that is SCNsusceptible. In embodiments, a mapping population may comprise about 10,about 20, about 30, about 40, about 50, about 60, about 70, about 80,about 90, about 95, about 100, about 150, about 200, about 250, about300, about 350, about 400, about 450, about 500, or more individuals. Insome embodiments, SCN resistant soybean germplasm 98860-71 may becrossed with one or more SCN susceptible germplasm(s) (e.g., 75213 and6CH026-035) to create mapping populations.

In some embodiments, the polymorphic markers may be single nucleotidepolymorphisms (SNPs) linked to or within the gene or QTL correspondingto the SCN resistance trait of interest. These SNP markers may bedetected by sequencing through the region containing the gene or QTLusing any DNA sequencing methods known in the art, including but notlimited to Sanger sequencing or high throughput sequencing (“NextGeneration”) methodologies that enable short or long sequence readsthrough the region of interest. In such embodiments, where genotyping bysequencing is used for the detection of SNP markers, primerscorresponding to the flanking sequences of the region containing theSNPs in gene or QTL of interest may be used for the sequencingchemistries in order to sequence through the region of interest. In suchembodiments, when different genotypes are used for sequencing throughthe region of interest for the detection of SNPs exemplified herein,other SNPs may be identified in addition to the SNPs exemplified herein.In such embodiments, the SNPs exemplified herein by themselves(individual SNPs) or in combination with other SNPs linked toexemplified sequences (haplotypes) may be utilized for differentiatinggenotypes towards marker assisted selection of plants for the SCNresistance trait of interest.

Primer Design and Linkage Screening.

Oligonucleotide probes (e.g., primers) may be designed to specificallydetect markers that are physically located in, near, or between QTLs andQTL intervals that correspond to a trait of interest. In general, anoligonucleotide probe may be designed that specifically hybridizes toonly one allele of a marker. In some embodiments, two oligonucleotideprobes are designed to detect an SNP marker, such that each specificallyhybridizes to the SNP allele to which the other probe does notspecifically hybridize. As is understood by those of skill in the art,the length or composition of oligonucleotide probes for a particularmarker may be varied according to established principles withoutrendering the probe non-specific for one allele of the marker.

In some embodiments, the oligonucleotide probes may be primers. Inspecific embodiments, primers may be designed to detect markers in aKASPAR™ genotyping assay. In particular embodiments, primers may bedesigned to detect markers linked to the SCN resistance phenotype insoybean using a KASPAR™ genotyping assay. In these and furtherembodiments, the detection system may provide a high-throughput andconvenient format for genotyping individuals in a mapping population,which may greatly facilitate the identification of individuals carryinga particular gene or trait, and may also greatly facilitate theimplementation or execution of a marker-assisted selection program.

In specific embodiments, the oligonucleotide probes may be primersdesigned to detect markers in a TAQMAN® genotyping assay. This methodutilizes primers specific to the marker closely linked to the SCNresistance gene and fluorescent labeled probes containing a singlenucleotide polymorphism (SNP). The SNP probe associated with resistanceis labeled with a fluorescent dye such as FAM while the probe associatedwith susceptibility is labeled with a different fluorescent dye such asVIC. The data is analyzed as the presence or absence of a fluorescentdye signal. The detection system may provide a high-throughput andconvenient format, such as multiplexing for genotyping individuals in amapping population, which may greatly facilitate the identification ofindividuals carrying a particular gene or trait, and may also greatlyfacilitate the implementation or execution of a marker-assistedselection program.

Additional markers may be identified as equivalent to any of theexemplary markers named herein (e.g., markers listed in Table 3, suchas, for example, rhgl-3995, BARC_010889_01691, and NCSB_004074), forexample, by determining the frequency of recombination between theexemplary marker and an additional marker. Such determinations mayutilize a method of orthogonal contrasts based on the method of Mather(1931), The Measurement of Linkage in Heredity, Methuen & Co., London,followed by a test of maximum likelihood to determine a recombinationfrequency. Allard (1956) Hilgardia 24:235-78. If the value of therecombination frequency is less than or equal to 0.10 (i.e., 10%), thenthe additional marker is considered equivalent to the particularexemplary marker for the purposes of use in the presently disclosedmethods.

Markers that are linked to any and all SCN resistance genes may beidentified in embodiments of the invention. Further, markers thatcontrol any and all of resistance contributing loci for all SCN HG racesmay be identified in embodiments of the invention.

A means for providing SCN resistance in soybean may be an SNP markerallele, the detection of which SNP marker allele in soybean plantsbelonging to, or derived from, germplasm 98860-71 provides at least astrong indication that the plant comprising the nucleic acid sequencehas the SCN resistance phenotype. In some examples, a means forproviding SCN resistance in soybean is a marker selected from the groupconsisting of the markers listed in Table 3. In particular examples, ameans for providing SCN resistance in soybean is a marker selected fromthe group consisting of rhgl-3995, BARC_010889 01691, and NCSB_004074.

A means for identifying soybean plants having the SCN resistancephenotype may be a molecule that presents a detectable signal when addedto a sample obtained from a soybean plant belonging to, or derived from,germplasm 98860-71 having the SCN resistance genotype, but which meansdoes not present a detectable signal when added to a sample obtainedfrom a soybean plant of belonging to, or derived from, germplasm98860-71 that does not have the SCN resistance phenotype. Specifichybridization of nucleic acids is a detectable signal, and a nucleicacid probe that specifically hybridizes to an SNP marker allele that islinked to the SCN resistance phenotype may therefore be a means foridentifying soybean plants having the SCN resistance phenotype. In someexamples, a means for identifying soybean plants having the SCNresistance phenotype is a probe that specifically hybridizes to a markerthat is linked to the SCN resistance phenotype.

C. Methods of Using Markers Linked to a Trait of Interest

Methods of using nucleic acid molecular markers that are linked to atrait of interest (e.g., SCN resistance in soybean) to identify plantshaving the trait of interest may result in a cost savings for plantdevelopers, because such methods may eliminate the need to phenotypeindividual plants generated during development (for example, by crossingsoybean plant varieties having SCN resistance with vulnerable plantvarieties).

In particular embodiments, markers linked to SCN resistance in soybeanmay be used to transfer segment(s) of DNA that contain one or moredeterminants of SCN resistance. In particular embodiments, the markersmay be selected from a group of markers comprising the markers listed inTable 3 and markers that are their equivalents. In some embodiments, amarker may be selected from the group consisting of rhgl-3995,BARC_010889 01691, and NCSB_004074. In some embodiments, a method forusing markers linked to SCN resistance in soybean to transfer segment(s)of DNA that contain one or more determinants of SCN resistance maycomprise analyzing the genomic DNA of two parent plants with probes thatare specifically hybridizable to markers linked to the SCN resistancephenotype; sexually crossing the two parental plant genotypes to obtaina progeny population, and analyzing those progeny for the presence ofthe markers linked to the SCN resistance phenotype; backcrossing theprogeny that contain the markers linked to the SCN resistance phenotypeto the recipient genotype to produce a first backcross population, andthen continuing with a backcrossing program until a final progeny isobtained that comprises any desired trait(s) exhibited by the parentgenotype and the SCN resistance phenotype. In particular embodiments,individual progeny obtained in each crossing and backcrossing step areselected by SCN marker analysis at each generation. In some embodiments,analysis of the genomic DNA of the two parent plants with probes thatare specifically hybridizable to markers linked to SCN resistancephenotype reveals that one of the parent plants comprises fewer of thelinked markers to which the probes specifically hybridize, or none ofthe linked markers to which the probes specifically hybridize. In someembodiments, individual progeny obtained in each cross and/or backcrossare selected by the sequence variation of individual plants.

In some embodiments, markers linked to the SCN resistance phenotype maybe used to introduce one or more determinants of SCN resistance into aplant (e.g., soybean) by genetic transformation. In particularembodiments, the markers may be selected from a group of markerscomprising the markers listed in Table 3 and markers that are theirequivalents. In some embodiments, a method for introducing one or moredeterminants of SCN resistance into a plant by genetic recombination maycomprise analyzing the genomic DNA of a plant (e.g., soybean) withprobes that are specifically hybridizable to markers linked to the SCNresistance phenotype to identify one or more determinants of SCNresistance in the plant; isolating a segment of the genomic DNA of theplant comprising the markers linked to the SCN resistance phenotype, forexample, by extracting the genomic DNA and digesting the genomic DNAwith one or more restriction endonuclease enzymes; optionally amplifyingthe isolated segment of DNA; introducing the isolated segment of DNAinto a cell or tissue of a host plant; and analyzing the DNA of the hostplant with probes that are specifically hybridizable to markers linkedto the SCN resistance phenotype to identify the one or more determinantsof SCN resistance in the host plant. In particular embodiments, theisolated segment of DNA may be introduced into the host plant such thatit is stably integrated into the genome of the host plant.

In some embodiments, markers that are linked to the SCN resistancephenotype may be used to introduce one or more determinants of SCNresistance into other organisms, for example, plants. In particularembodiments, the markers can be selected from a group of markers listedin Table 3 and markers that are their equivalents. In some embodiments,a method for introducing one or more determinants of SCN resistance intoan organism other than soybean may comprise analyzing the genomic DNA ofa plant (e.g., a soybean plant) with probes that are specificallyhybridizable to markers linked to the SCN resistance phenotype toidentify one or more determinants of SCN resistance in the plant;isolating a segment of the genomic DNA of the plant comprising the oneor more determinants of SCN resistance, for example, by extracting thegenomic DNA and digesting the genomic DNA with one or more restrictionendonuclease enzymes; optionally amplifying the isolated segment of DNA;introducing the isolated segment of DNA into an organism other thansoybean; and analyzing the DNA of the organism other than soybean withprobes that are specifically hybridizable to markers linked to the SCNresistance phenotype to identify the one or more determinants of SCNresistance in the organism. In other embodiments, the isolated segmentof DNA may be introduced into the organism such that it is stablyintegrated into the genome of the organism.

In some embodiments, markers that are linked to the SCN resistancephenotype may be used to identify a plant with one or more determinantsof SCN resistance. In some embodiments, the plant may be a soybeanplant. For example, the plant may be a soybean plant of germplasm98860-71. In particular embodiments, nucleic acid molecules (e.g.,genomic DNA or mRNA) may be extracted from a plant. The extractednucleic acid molecules may then be contacted with one or more probesthat are specifically hybridizable to markers linked to the SCNresistance phenotype. Specific hybridization of the one or more probesto the extracted nucleic acid molecules is indicative of the presence ofone or more determinants of SCN resistance in the plant.

In some embodiments, markers that are linked to multiple determinants ofSCN resistance may be used simultaneously. In other embodiments, markersthat are linked to only one determinant of SCN resistance may be used.In specific examples, markers that are linked to SCN resistance withrespect to one or more particular SCN HG races (e.g., race 1, race 2,race 3, race 5, and race 14) may be used simultaneously For example, aplurality of markers that are linked to SCN resistance with respect todifferent SCN HG races may be used simultaneously.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Materials and Methods

24 soybean cultivars and SCN mapping parents were used to identifymarkers linked to the SCN resistance phenotype. 14 of the cultivars wereSCN susceptible: 75110, 75155, 75163, 99630, 99726, 95895-755PRU,99345-31, 75192, 75209, 75159, Essex, Williams82, 75213, and 6CH026-035.10 of the cultivars were SCN resistant: Maverick, Peking, PI209332,PI437654, 99811, 99294, Forrest, PI88788, PI437654, and 98860-71.

SCN bioassay: SCN bioassays were performed to generate phenotypeinformation of mapping populations produced by crossing SCN resistantsoybean variety 98860-71 with SCN susceptible soybean varieties 75213and 6CH026-035. The phenotype information of the mapping population usedis listed in Table 3. The industry does not have a uniform approach tocategorizing resistance levels in soybean varieties. Therefore,resistance levels were categorized in terms of “SCN score:” SCN score0-10=R (resistance); SCN score 10.1-29.9=MR (medium resistance); SCNscore 30.0-59.9=MS (medium susceptible); and 60+=S (susceptible). SCNindex values were determined by comparing testing lines to knownsusceptible and resistant lines. The index score was directly based onthe percentage of SCN susceptibility observed for the sample. Forexample, if a testing line had 10 cysts on each of 9 plants, andWilliams (susceptible) had 100 cysts on each of 9, then the testing linewas categorized with an index of 10%. The final index was the average ofthe scores of the 9 plants.

KASPAR™ reactions: KASPAR™ primers were designed using PRIMERPICKER™tool in KLIIVIS™ (KBioscience Laboratory Management System) by providingDNA sequences with SNPs. Three primers, A1 (Allele specific primer 1),A2 (Allele specific primer 2), and C (common reverse primer) weredesigned for each SNP sequence based on KASPAR™ chemistry. An assay mixof each KASPAR™ reaction was prepared as in the KASPAR™ SNP GenotypingSystem v2.0. The final reaction volume was 5 μL per reaction, including1 μL DNA template (5 ng/μL), 2.5 μL 2× Reaction Mix, 0.06875 μL Assaymix, 0.04 μL 50 mM MgCl₂, and 1.39125 μL ddH₂O. The assay was carriedout in 384-well format. The thermocycle conditions used during the assaywere according to the manufacturer's instructions: 94° C. for 15minutes; 20 cycles of 94° C. for 10 seconds, 57° C. for 5 seconds, and72° C. for 10 seconds; and 22 cycles of 94° C. for 10 seconds, 57° C.for 20 seconds, and 72° C. for 40 seconds. PCR plates were centrifuged,and allele-specific FAM and VIC intensities were read on aspectrofluorometer (Tecan GENIOS™, Männedorf, Switzerland) at roomtemperature. Data were directly loaded and analyzed on KLIMS™ usingKLUSTER CALLER™.

Example 2 Identification of Physical Positions of QTLs and QTL Intervalsthat are Linked to SCN Resistance Genes

QTLs that are involved in SCN resistance were initially identified bystudying the SCN literature. The initially identified SCN-associatedQTLs found in the SCN literature are listed in FIGS. 1a and 1 b.

From the list of QTLs that were initially identified in the SCNliterature, several distinct QTL intervals that are involved inresistance to different SCN races were determined by reference to thesoybean genome map. See, e.g., FIG. 2. For example, QTL intervals onlinkage group (LG) G were determined as shown in FIG. 3; QTL intervalson LG A2 were determined as shown in FIG. 4; QTL intervals on LG Bi weredetermined as shown in FIG. 5; and QTL intervals on LG I were determinedas shown in FIG. 6. Table 2 lists exemplary QTLs and their correspondingdetermined QTL intervals that are associated with resistance todifferent SCN races.

Example 3 Identification of SNP Markers that are Physically LocatedIn/Near/Between the QTLs and QTL Intervals that are Linked to SCNResistance Genes

The soybean genome was searched using BLAST™ for SNP markers that arephysically located in, near, or between the QTL intervals that weredetermined. It was hypothesized that some of these SNP markers may belinked to the SCN resistance phenotype. A total of 79 SNP markers wereselected for an initial screen using 24 soybean lines (14 SCNsusceptible and 10 SCN resistant) to determine which, if any, of theseSNP markers are linked to the SCN resistance phenotype. 25 of the 79markers were located on LG G, 12 of the markers were located on LG A₂,22 of the markers were located on LG Bi, and 20 of the markers werelocated on LG I. All of the 79 selected markers are listed in Table 2.

TABLE 2 List of the 79 SNPs for screening with 24 soybean lines MarkerSNP allele Linkage group Chromosome BARC_018419_02911 [C/T] A2 8BARC_025811_05088 [C/T] A2 8 BARC_040339_07714 [A/G] A2 8 NCSB_001710[A/T] A2 8 NCSB_001716 [T/C] A2 8 NCSB_001717 [A/C] A2 8 NCSB_001718[A/G] A2 8 NCSB_001719 [A/C] A2 8 BARC_007704_00081 [T/A] B1 11BARC_010169_00537 [C/T] B1 11 BARC_013547_01157 [A/T] B1 11BARC_018557_03202 [A/G] B1 11 BARC_018649_03221 [C/T] B1 11BARC_025703_04999 [C/G] B1 11 BARC_035379_07178 [G/T] B1 11BARC_904050_01007 [A/T] B1 11 NCSB_002644 [A/G] B1 11 NCSB_002645 [A/G]B1 11 NCSB_002646 [A/G] B1 11 NCSB_002647 [A/T] B1 11 NCSB_002648 [T/G]B1 11 NCSB_002649 [T/C] B1 11 NCSB_002650 [C/G] B1 11 NCSB_002651 [T/C]B1 11 NCSB_002652 [T/C] B1 11 NCSB_002653 [A/C] B1 11 NCSB_002654 [A/C]B1 11 NCSB_002655 [A/C] B1 11 NCSB_002656 [A/G} B1 11 NCSB_002657 [A/C]B1 11 BARC_003180_00257 [C/T] G 18 BARC_010889_01691 [C/T] G 18BARC_012237_01755 [A/C] G 18 BARC_015377_01829 [A/C] G 18BARC_027452_06569 [A/T] G 18 BARC_028299_05817 [C/G] G 18BARC_035305_07162 [A/T] G 18 BARC_G01475_00237 [A/C] G 18 NCSB_004072[A/G] G 18 NCSB_004073 [A/G] G 18 NCSB_004074 [A/C] G 18 NCSB_004078[A/G] G 18 NCSB_004079 [C/G] G 18 NCSB_004080 [C/G] G 18 NCSB_004081[A/G] G 18 NCSB_004082 [T/C] G 18 NCSB_004083 [A/G] G 18 NCSB_004084[A/T] G 18 NCSB_004085 [A/T] G 18 NCSB_004086 [A/T] G 18 NCSB_004097[T/C] G 18 NCSB_004098 [T/C] G 18 NCSB_004107 [T/G] G 18 NCSB_004108[A/C] G 18 NCSB_004109 [C/G] G 18 rhg1_2564 [G/—] G 18 rhg1_3995 [A/C] G18 rhg1_689 [A/C] G 18 rhg1_757 [T/C] G 18 NCSB_004874 [A/G] I 20NCSB_004875 [T/G] I 20 NCSB_004877 [A/T] I 20 NCSB_004879 [A/G] I 20NCSB_004882 [A/T] I 20 NCSB_004883 [A/G] I 20 NCSB_004884 [T/G] I 20NCSB_004886 [T/C] I 20 NCSB_004887 [T/G] I 20 NCSB_004889 [A/G] I 20NCSB_004890 [T/C] I 20 NCSB_004891 [T/C] I 20 NCSB_004893 [T/C] I 20NCSB_004894 [T/C] I 20 NCSB_004895 [A/G] I 20 NCSB_004897 [T/C] I 20NCSB_004898 [A/T] I 20 NCSB_004899 [A/C] I 20 NCSB_004900 [A/T] I 20NCSB_004903 [T/G] I 20

Example 4 KASPAR™ Assay Development

Initial screening of the 79 SNP markers in the parental 24 lines wasperformed using KASPAR™ genotyping assays. 75 of the SNP markers werevalidated.

21 SNP markers on LG G (Gm 18) were validated: NCSB_004072, BARC_01527701929, NCSB_004073, NCSB_004074, NCSB_004075, NCSB_004076, NCSB_004077,NCSB_004078, NCSB_004079, NCSB_004080, NCSB_004081, NCSB_004082,NCSB_004083, NCSB_004084, NCSB_004085, NCSB_004086, NCSB_004097,NCSB_004098, NCSB_004107, NCSB_004108, and NCSB_004109.

12 SNP markers on LG A2 (Gm 08) were validated: BARC_025911_05089, BARC019419_02921, NCSB_001710, NCSB_001711, NCSB_001712, NCSB_001713,NCSB_001714, NCSB_001715, NCSB_001716, NCSB_001717, NCSB_001718, andNCSB_001719.

22 SNP markers on LG B1 (Gm 11) were validated: NCSB_002644,NCSB_002645, NCSB_002646, NCSB_002647, NCSB_002648, NCSB_002649,NCSB_002650, NCSB_002651, NCSB_002652, NCSB_002653, NCSB_002654,NCSB_002655, NCSB_002656, NCSB_002657, BARC 007704_00091, BARC010269_00537, BARC 904050_01007, BARC 019557_03202, BARC 018649_03221,BARC 025703_04999, BARC 013547_01157, and BARC_035379_07178.

20 SNP markers on LG I (Gm 20) were validated: NCSB_004974, NCSB_004975,NCSB_004977, NCSB_004979, NCSB_004882, NCSB_004883, NCSB_004884,NCSB_004886, NCSB_004887, NCSB_004889, NCSB_004880, NCSB_004882,NCSB_004883, NCSB_004884, NCSB_004885, NCSB_004887, NCSB_004888,NCSB_004899, NCSB_004900, and NCSB_004903 .

The initial screening identified 44 of the SNP markers as polymorphicamong the 24 parental lines. 24 polymorphic markers were selected(NCSB_001716 (LG A2), NCSB_002645 (B₁), NCSB_002646 (B₁), NCSB_002648(B₁), NCSB_002651 (B₁), NCSB_002652 (B₁), NCSB_002654 (B₁), NCSB_002656(B₁), BARC 013547 01157 (B₁), NCSB_004073 (G), NCSB_004074 (G),NCSB_004078 (G), NCSB_004080 (G), NCSB_004084 (G), NCSB_004085 (G),NCSB_004097 (G), NCSB_004109 (G), BARC 012237 01755 (G), rghl-689 (G),rgh1-757 (G), rgh1-2564 (G), rgh1-3995 (G), and NCSB_004900 (I)) forfurther linkage testing with mapping populations produced by crossingSCN resistant soybean variety 98860-71 with SCN susceptible soybeanvarieties 75213 and 6CH026-035. FIG. 7 shows representative genotypingdata from the KASPAR™ assay for four of the polymorphic markers.

15 of the SNP markers were polymorphic between SCN resistant andsusceptible parents. These 15 polymorphic SNP markers were subsequentlyscreened against 93 individuals in the mapping populations.

Of the fifteen SNP markers that were polymorphic among the parentallines that were tested against individuals of the mapping population,only three SNPs showed co-segregation with the SCN resistance trait:NCSB_004074, BARC_010889_01691, and rhg1-3995. FIG. 8. The KASPAR™primer sequences that were used to genotype individuals for these threemarkers are listed in Table 3.

TABLE 3 The KASPAR ™ primer sequences of SNP markers rhg1-GAAGGTGACCAAGTTCATGCTGGAATTATGTTGGGTTTT 3995A1 TTTTCTTTCTGT(SEQ ID NO: 1) rhg1- GAAGGTCGGAGTCAACGGATTGAATTATGTTGGGTTTTT 3995A2TTTCTTTCTGG (SEQ ID NO: 2) rhg1- GCCCAGAAAAAAGGGATAAATAACGGATA 3995C1(SEQ ID NO: 3) NCSB_ GAAGGTGACCAAGTTCATGCTATTATGTTGTAACACAAA 004074A1TTTGCACCTCAT (SEQ ID NO: 4) NCSB_GAAGGTCGGAGTCAACGGATTATGTTGTAACACAAATTT 004074A2 GCACCTCAG(SEQ ID NO: 5) NCSB_ CTATACAACTAAATCGTAATTCCATTGTAT 004074C1(SEQ ID NO: 6) BARC_ GAAGGTGACCAAGTTCATGCTGAAAAAATAAAATTGATC 010889-ATCACATATGGTTAG 0169 (SEQ ID NO: 7) 1A1 BARC_GAAGGTCGGAGTCAACGGATTGAAAAAATAAAATTGATC 010889- ATCACATATGGTTAA 0169(SEQ ID NO: 8) 1A2 BARC_ TAAGTGAGGGCAATGTATTAGTATYAAGTA 010889-(SEQ ID NO: 9) 0169 1C1

Using the genome nucleic acid sequence of soybean cultivar Williams 82as a reference, BARC 010889 01691 is located on chromosome 18 at1,674,511 bp; NCSB_004074 is located on chromosome 18 at 1,663,671 bp;and rghl-3995 is located on chromosome 18 at 1,714,741 bp. All threelinked markers (NSCB_004074, BARC_010889_01691, and rhg1-3995) arelocated either within the rhg1 locus (rhg1-3995), or close to it onlinkage group G (BARC_010889_01691 and NCSB_004074).

For the resistant and medium resistant phenotypes, all three linkedmarker genotypes were congruent with the phenotype. With respect to thesusceptible lines, BARC_010889_01691 had 5 mismatches with phenotypes,NSCB_004074 had 9 mismatches, and rhg1-3995 had 6 mismatches. See Table4.

TABLE 4 Comparison of the phenotype score and genotype scores of 93lines derived from two mapping populations plus 3 parents. SCN SCNSample Resistance Score rhg1_3995 NCSB_004074 BARC_010889_01691 40779 R1.00 C:C A:A T:T 40785 R 1.60 C:C A:A T:T 29110 R 2.30 C:C A:A T:T 40780R 2.30 C:C A:A T:T 29148 R 2.50 C:C A:A T:T 40781 R 2.50 C:C A:A T:T40799 R 2.50 C:C A:A T:T 29226 R 3.10 C:C A:A T:T 40910 R 4.00 C:C A:AT:T 29040 R 4.10 C:A A:A T:T 40908 R 4.50 C:C A:A T:T 19152 R 4.70 C:CA:A T:T 29149 R 4.80 C:C A:A T:T 29023 R 5.60 C:C A:A T:T 40907 R 5.80C:C A:A T:T 40959 R 6.10 C:C A:A T:T 29181 R 6.50 C:C A:A T:T 29151 R6.60 C:C A:A T:T 19209 R 6.90 C:C A:A T:T 40989 R 7.10 C:C A:A T:T 29189R 7.60 C:C A:A T:T 21692 R 8.30 C:C A:A T:T 29089 R 8.80 C:C A:A T:T21553 R 9.00 C:C A:A T:T 40833 R 9.20 C:C A:A T:T 21642 R 9.60 C:C A:AT:T 29228 R 9.70 C:C A:A T:T 40957 R 9.80 C:C A:A T:T 29191 MR 10.2 C:CA:A T:T 40935 MR 10.7 C:C A:A T:T 98860-71(P1-R) R/MR 11.3 C:C A:A T:T21648 R/MR 11.8 C:C A:A T:T 19155 MR 12.4 C:C A:A T:T 40808 MR 12.6 C:CA:A T:T 40940 MR 13.0 C:C A:A T:T 40831 MR 13.7 C:C A:A T:T 40932 MR14.5 C:C A:A T:T 29180 MR 16.9 C:C A:A T:T 40937 MR 17.9 C:C A:A T:T40958 MR 19.6 C:C A:A T:T 40936 MR 21.5 C:C A:A T:T 41022 X 22.7 C:C A:AT:T 19182 MR 23.3 C:C A:A T:T 40810 X 27.5 C:C A:A T:T 41015 X 27.6 C:CA:A T:T 29039 MS 31.70 A:A C:C C:C 29212 MS 37.50 C:C A:A T:T 40905 X39.70 A:A A:A C:C 40834 X 46.50 A:A C:C C:C 40906 MS 46.60 A:A A:A C:C41016 MS 46.80 A:A C:C C:C 29179 MS 48.10 A:A C:C C:C 29119 MS 59.50 A:AC:C C:C 29021 MS 59.80 A:A C:C C:C 29142 S 61.90 A:A C:C C:C 40939 S66.40 A:A C:C C:C 41026 S 67.90 C:C C:C C:C 40909 S 68.90 A:A C:C C:C29150 S 70.60 A:A C:C C:C 40942 S 71.20 A:A A:A C:C 40711 S 71.50 C:CA:A T:T 40931 S 73.80 A:A C:C C:C 21633 S 75.00 C:C A:A T:T 29190 S79.70 A:A C:C C:C 29229 S 79.90 A:A C:C C:C 40938 S 80.10 A:A C:C C:C40873 X 81.50 C:C A:A T:T 29222 S 81.90 A:A C:C C:C 40941 S 84.00 A:AC:C C:C 40934 S 85.70 A:A C:C C:C 29376 S 88.90 A:A C:C C:C 75213(P2-S)S 88.90 A:A C:C C:C 40990 S 90.10 A:A C:C C:C 29224 S 90.60 A:A C:C C:C29399 S 93.50 A:A C:C C:C 40798 X 96.40 A:A A:A C:C 21693 X 98.00 A:AC:C C:C 21684 X 98.10 A:A C:C C:C 6CH026-035 S 101.20 A:A C:C C:C (P3-S)41014 S 109.40 A:A C:C C:C 41027 S 125.60 A:A C:C C:C 29514 S 130.00 A:AC:C C:C 21688 S 133.90 A:A C:C C:C 40992 S 134.40 A:A C:C C:C 21700 S134.60 A:A C:C C:C 40991 S 138.80 A:A C:C C:C 40782 S 147.00 A:A C:C C:C29639 S 163.20 A:A C:C C:C 40783 S 178.20 C:C A:A T:T 40778 S 187.40 A:AC:C C:C 21683 S 204.40 A:A C:C C:C 40809 S 207.70 A:A C:C C:C 40835 S212.90 A:A C:C C:C 40832 S 215.20 A:A C:C C:C 21694 S 254.90 A:A C:C C:C21698 S 380.00 A:A C:C C:C R = resistance; MR = medium resistance; S =susceptible; MS = medium susceptible; and X = inconsistency.

Once we have identified any susceptible genotypes with these 3 SNPmarkers, there is a 0% false negative rate. In other words, we canidentify with perfect accuracy the SCN susceptible phenotype using the 3markers. We also can predict the SCN resistant genotype with a “falsepositive” rate of about 10-18% (5 or 9 divided by 51, the total numberof susceptible samples). Therefore, of the SCN resistant genotypesidentified, only 5-9% of them would be expected to exhibit a SCNsusceptible phenotype.

Example 5 SNP markers in LG A₂, LG Bi, and LG I that are Linked to theSCN Resistance Phenotype

The soybean genome is searched using BLAST™ for SNP markers that arephysically located in, near, or between QTL intervals associated withSCN resistance on linkage groups Az, Bi, and I. A list of SNP markers isproduced by the BLAST™ search. A plurality of SNP markers are selectedfor an initial screen using SCN susceptible and SCN resistant soybeanlines to determine which, if any, of these SNP markers in linkage groupsA₂, B₁, and I are linked to the SCN resistance phenotype.

Initial screening of the selected SNP markers in the parental lines isperformed using KASPAR™ genotyping assays. A set of the selected SNPmarkers are validated, a subset of which are identified as polymorphicamong the parental lines. At least one of the polymorphic SNP markersis/are used for linkage testing with mapping populations produced bycrossing an SCN resistant soybean variety with one or more SCNsusceptible soybean varieties. One or more of these polymorphic SNPmarkers are screened against individuals in the mapping populations.

SNPs that co-segregate with the SCN resistance trait in individuals ofthe mapping populations are identified as markers on linkage groups A₂,B₁, and I that are linked to SCN resistance in the SCN resistant parentvariety. The linked marker genotypes match the phenotypes observed inthe individuals of the mapping population.

Example 6 SNP Markers Linked to the SCN Resistance Phenotype inGermplasm JTN-5109

A plurality of SNP markers that are physically located in, near, orbetween QTL intervals associated with SCN resistance (for example, SNPmarkers selected from the group of markers listed in Table 3) areselected for an initial screen using SCN resistant soybean varietyJTN-5109 and SCN susceptible soybean lines to determine which, if any,of these SNP markers are linked to the SCN resistance phenotype insoybean variety JTN-5109.

Initial screening of the selected SNP markers in the parental lines isperformed using KASPAR™ genotyping assays. A set of the selected SNPmarkers are validated, a subset of which are identified as polymorphicamong soybean variety JTN-5109 and the SCN susceptible parental lines.At least one of the polymorphic SNP markers is/are used for linkagetesting with mapping populations produced by crossing soybean varietyJTN-5109 with one or more SCN susceptible soybean varieties. These oneor more polymorphic SNP markers are screened against individuals in themapping populations.

SNPs that co-segregate with the SCN resistance trait in individuals ofthe mapping populations are identified as markers that are linked to SCNresistance in soybean variety JTN-5109. The linked marker genotypesmatch the phenotypes observed in the individuals of the mappingpopulation.

Example 7 SNP Markers that are Linked to the SCN Resistance Phenotype inHG Races Other Than Race 3

Mapping populations are developed specifically for an HG race other thanrace 3 by crossing an SCN resistant soybean variety selected from thegroup consisting of PI 88788, Peking, PI 437654, PI 90763, PI 438489B,PI 89772, PI209332, PUSCN14, Hartwig, Forrest, and Pyramid with one ormore SCN susceptible soybean varieties.

The soybean genome is searched using BLAST™ for SNP markers that arephysically located in, near, or between QTL intervals associated withSCN resistance with respect to the specific HG race. A list of SNPmarkers is produced by the BLAST™ search. A plurality of SNP markers areselected for an initial screen using the selected SCN resistant soybeanvariety and SCN susceptible varieties to determine which, if any, of theSNP markers are linked to the SCN resistance phenotype with respect tothe specific HG race.

Initial screening of the selected SNP markers in the parental lines isperformed using KASPAR™ genotyping assays. A set of the selected SNPmarkers are validated, a subset of which are identified as polymorphicamong the parental lines. At least one of the polymorphic SNP markersis/are used for linkage testing with mapping populations produced bycrossing the selected SCN resistant soybean variety with one or more SCNsusceptible soybean varieties. These one or more polymorphic SNP markersare screened against individuals in the mapping populations.

SNPs that co-segregate with the SCN resistance trait in individuals ofthe mapping populations are identified as markers that are linked to SCNresistance in the SCN resistant parent variety with respect to thespecific HG race. The linked marker genotypes match the phenotypesobserved in the individuals of the mapping population.

1. An oligonucleotide that is specifically hybridizable to a markerlinked to the SCN resistance phenotype in soybean, wherein theoligonucleotide is: an oligonucleotide that binds under very highstringency conditions to a reference polynucleotide or the perfectcomplement of the reference polynucleotide, wherein the referencepolynucleotide consists of SEQ ID NO:11 comprising a thymine (T) atnucleotide position 98 of the reference polynucleotide, wherein theoligonucleotide does not bind under the very high stringencyhybridization conditions to the reference polynucleotide comprising acytosine (C) at nucleotide position 98 of the reference polynucleotide;or an oligonucleotide that binds under very high stringencyhybridization conditions to a second reference polynucleotide or theperfect complement of the second reference polynucleotide, wherein thesecond reference polynucleotide consists of SEQ ID NO:12 comprising acytosine (C) at nucleotide position 214 of the second referencepolynucleotide, wherein the oligonucleotide does not bind under the veryhigh stringency hybridization conditions to the second referencepolynucleotide comprising an adenine (A) at nucleotide position 214 ofthe second reference polynucleotide.
 2. The oligonucleotide of claim 1,wherein the oligonucleotide is between 19 and 30 nucleotides in length.3. The oligonucleotide of claim 1, comprising a label that is afluorophore or ³²P.
 4. The oligonucleotide of claim 1, wherein theoligonucleotide binds under very high stringency conditions to thereference polynucleotide or the perfect complement of the referencepolynucleotide, wherein the reference polynucleotide consists of SEQ IDNO:11 comprising a thymine (T) at nucleotide position 98 of thereference polynucleotide, wherein the oligonucleotide does not bindunder the very high stringency hybridization conditions to the referencepolynucleotide comprising a cytosine (C) at nucleotide position 98 ofthe reference polynucleotide.
 5. The oligonucleotide of claim 1, whereinthe oligonucleotide binds under very high stringency hybridizationconditions to the second reference polynucleotide or the perfectcomplement of the second reference polynucleotide, wherein the secondreference polynucleotide consists of SEQ ID NO:12 comprising a cytosine(C) at nucleotide position 214 of the second reference polynucleotide,wherein the oligonucleotide does not bind under the very high stringencyhybridization conditions to the second reference polynucleotidecomprising an adenine (A) at nucleotide position 214 of the secondreference polynucleotide.
 6. A method for producing an SCN-resistantsoybean plant, the method comprising: crossing a first soybean planthaving the trait of SCN resistance with a second soybean plant that doesnot have the trait of SCN resistance, to produce F₁ soybean plants; andidentifying an F₁ soybean plant comprising genomic DNA that binds theoligonucleotide of claim 1 under very high stringency hybridizationconditions.
 7. The method according to claim 6, the method furthercomprising harvesting seed from the identified F₁ soybean plant.
 8. Themethod according to claim 6, wherein the oligonucleotide binds undervery high stringency conditions to the reference polynucleotide or theperfect complement of the reference polynucleotide, wherein thereference polynucleotide consists of SEQ ID NO:11 comprising a thymine(T) at nucleotide position 98 of the reference polynucleotide, whereinthe oligonucleotide does not bind under the very high stringencyhybridization conditions to the reference polynucleotide comprising acytosine (C) at nucleotide position 98 of the reference polynucleotide.9. The method according to claim 6, wherein the oligonucleotide bindsunder very high stringency hybridization conditions to the secondreference polynucleotide or the perfect complement of the secondreference polynucleotide, wherein the second reference polynucleotideconsists of SEQ ID NO:12 comprising a cytosine (C) at nucleotideposition 214 of the second reference polynucleotide, wherein theoligonucleotide does not bind under the very high stringencyhybridization conditions to the second reference polynucleotidecomprising an adenine (A) at nucleotide position 214 of the secondreference polynucleotide.
 10. A method for introgressing a determinantof SCN resistance into a soybean variety, the method comprising: (a)sexually crossing a first parental plant having a donor genotypecomprising the determinant of SCN resistance with a second parentalplant having a recipient genotype to obtain a progeny plant population;(b) identifying from the progeny plant population a plant with genomicDNA that binds the oligonucleotide of claim 1 under very high stringencyhybridization conditions, thereby determining the presence of thedeterminant of SCN resistance in the plant; (c) backcrossing theidentified plant from the progeny population that comprises thedeterminant of SCN resistance to the recipient genotype to produce anext generation population; (d) determining if a member of the nextgeneration population comprises genomic DNA that binds theoligonucleotide under very high stringency hybridization conditions anda desired trait from the recipient genotype; and (e) if no member of thenext generation population comprises genomic DNA that binds theoligonucleotide under very high stringency hybridization conditions andthe desired trait from the recipient genotype, repeating steps (c) and(d) until an individual is identified that comprises genomic DNA thatbinds the oligonucleotide under very high stringency hybridizationconditions and the desired trait from the recipient genotype.
 11. Themethod according to claim 10, wherein the oligonucleotide binds undervery high stringency conditions to the reference polynucleotide or theperfect complement of the reference polynucleotide, wherein thereference polynucleotide consists of SEQ ID NO:11 comprising a thymine(T) at nucleotide position 98 of the reference polynucleotide, whereinthe oligonucleotide does not bind under the very high stringencyhybridization conditions to the reference polynucleotide comprising acytosine (C) at nucleotide position 98 of the reference polynucleotide.12. The method according to claim 10, wherein the oligonucleotide bindsunder very high stringency hybridization conditions to the secondreference polynucleotide or the perfect complement of the secondreference polynucleotide, wherein the second reference polynucleotideconsists of SEQ ID NO:12 comprising a cytosine (C) at nucleotideposition 214 of the second reference polynucleotide, wherein theoligonucleotide does not bind under the very high stringencyhybridization conditions to the second reference polynucleotidecomprising an adenine (A) at nucleotide position 214 of the secondreference polynucleotide.
 13. A method for introgressing a determinantof SCN resistance into a soybean variety, the method comprising: (a)sexually crossing a first parental plant having a donor genotype with asecond parental plant having a recipient genotype comprising thedeterminant of SCN resistance to obtain a progeny plant population; (b)identifying from the progeny plant population a plant with genomic DNAthat binds the oligonucleotide of claim 1 under very high stringencyhybridization conditions, thereby determining the presence of thedeterminant of SCN resistance in the plant; (c) backcrossing theidentified plant from the progeny population that comprises thedeterminant of SCN resistance to the recipient genotype to produce anext generation population; (d) determining if a member of the nextgeneration population comprises genomic DNA that binds theoligonucleotide under very high stringency hybridization conditions anda desired trait from the donor genotype; and (e) if no member of thenext generation population comprises genomic DNA that binds theoligonucleotide under very high stringency hybridization conditions andthe desired trait from the donor genotype, repeating steps (c) and (d)until an individual is identified that comprises genomic DNA that bindsthe oligonucleotide under very high stringency hybridization conditionsand the desired trait from the donor genotype.
 14. The method accordingto claim 13, wherein the oligonucleotide binds under very highstringency conditions to the reference polynucleotide or the perfectcomplement of the reference polynucleotide, wherein the referencepolynucleotide consists of SEQ ID NO:11 comprising a thymine (T) atnucleotide position 98 of the reference polynucleotide, wherein theoligonucleotide does not bind under the very high stringencyhybridization conditions to the reference polynucleotide comprising acytosine (C) at nucleotide position 98 of the reference polynucleotide.15. The method according to claim 13, wherein the oligonucleotide bindsunder very high stringency hybridization conditions to the secondreference polynucleotide or the perfect complement of the secondreference polynucleotide, wherein the second reference polynucleotideconsists of SEQ ID NO:12 comprising a cytosine (C) at nucleotideposition 214 of the second reference polynucleotide, wherein theoligonucleotide does not bind under the very high stringencyhybridization conditions to the second reference polynucleotidecomprising an adenine (A) at nucleotide position 214 of the secondreference polynucleotide.